Conjugates formed from heat shock proteins and oligo-or polysaccharides

ABSTRACT

The present invention provides conjugate compounds comprising at least one heat shock protein or portion thereof including at least one immunostimulatory domain and at least one capsular oligosaccharide or polysaccharide of a pathogenic bacteria. The compound comprises oligosaccharides of the Meningococci C (MenC) group and a heat shock protein selected from  M. bovis  BCG GroE1-type 65 kDa hsp (hspR65), recombinant  M. tuberculosis  DnaK-type 70 kDa hsp (hspR70) and a heat shock protein from  H. pylori . The invention also provides processes for producing conjugate compounds, pharmaceutical compositions comprising conjugate compounds, therapeutic compositions comprising conjugate compounds, and methods of inducing an immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase of international applicationPCT/EP93/00516, filed Mar. 8, 1993. This application claims priorityfrom application F192A000058, filed Mar. 6, 1992.

FIELD OF THE INVENTION

The present invention relates to conjugated compounds consisting of heatshock proteins and polysaccharides or oligosaccharides, in particularthose polysacccharides or oligosaccharides derived from the capsule ofpathogenic microorganisms. Such compounds are capable of inducing theformation of anti-polysaccharide antibodies and are accordingly usefulas vaccines for use in man and in animals.

STATE OF THE ART

Bacteria are the aetiological agents for a wide range of diseaseconditions.

Examples of such diseases include meningitis caused by Neisseriameningitidis and other infections caused by Haemophilus influenzae Typeb (Hib) or Streptococcus (including Pneumococcus), typhoid fever causedby infection with Salmonella typhi, intestinal disease caused bynon-typoidal Salmonella or Shigella bacteria.

It is known that protective immunity to capsular bacteria is mediated byantibodies to the capsular polysaccharides. It is also known that, inorder to obtain sufficient stimulation of the immune system, it isnecessary to conjugate capsular polysaccharides to carrier proteins(Robbins et al, J. Infect. Dis., 1990, 161,821-832).

In particular, there have been described in the literature conjugatedcompounds consisting of polysaccharides (for example Group Cmeningococcal polysaccharide (MenC), Hib and Group A meningococcalpolysaccharide (MenA)) and proteins such as CRM-197 (a peptide derivedfrom Corynebacterium diphtheriae), TD (Diphtheria toxoid) or TT (Tetanustoxoid—see Peeters et al. Inf.Immun., (October 1991), 3504-3510;Claesson et al., J. Pediatrics St Louis, 112(5), 695-702, (May 1988).

Some such vaccines are already used with good results in clinicalpractice. However, there exists the need to identify novel proteincarriers which impart to the conjugates immunogenic properties betterthan those achieved with the carriers used hitherto.

The present invention relates to the use of heat shock proteins as aprotein carrier to increase the immunogenic response of oligosaccharidesand polysaccharides.

Heat shock proteins are known to contain a significant number of Tepitopes and thus to stimulate the cellular immune system.

A conjugated compound of the heat shock protein of Mycobacterium bovis(65 kDa), as a carrier for a malarial epitope, has been described asinducing a marked immunity in animals pre-immunised with BacillusCalmette-Guérin (BCG) without requiring adjuvants (Lussow et al Eur. J.Immunol., 1991, 21,2297-2302). It is however to be noted that theeffects observed in Lussow et al relate to T cell dependent effectsexhibited by peptides (which are well known to be T-cell dependent)conjugated to heat shock proteins.

More particularly, because the heat shock proteins are well conservedacross bacteria of different strains and type, adventitious infectionwith bacteria, which is a continuous process, will ensure that theimmune system remains sensitised to heat shock proteins, thus ensuring agood response to the conjugate compounds of the invention either atprimary vaccination or on administration of a booster vaccination.

The present invention permits the use of bacterial capsularpolysaccharides and oligosaccharides to be used without adjuvants(although adjuvants can be used).

Large numbers of children are given BCG vaccine (which will includebacterial heat shock proteins) to guard against tuberculosis andtherefore a conjugate of the present invention containing a heat shockprotein as the carrier would find a large number of subjects alreadypre-immunised with the carrier precisely as a result of the BCGvaccination which they have undergone.

Again, since the heat shock proteins are highly conserved even thepopulation which has not been vaccinated with BCG can easily developimmunity (as a result of natural interaction with other bacteria) andcan hence find itself in a state of being able to develop a good immuneresponse following vaccination with a conjugate composed of a heat shockprotein and a T cell-independent antigen (oligosaccharide orpolysaccharide). Thus the carriers of the present invention uniquelyexploit the high conservation of heat shock proteins across bacteria andT-cell memory to ensure high titre vaccination.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a conjugatecompound comprising at least one heat shock protein or portion thereofincluding at least one immunostimulatory domain and at least oneoligosaccharide or polysaccharide.

The heat shock protein may be any heat shock protein capable ofexhibiting an immunostimulatory effect in animals, preferably humans.

The heat shock proteins are highly conserved in bacteria, parasites andmammals. Any heat shock protein can be used in the conjugates of thepresent invention, provided it exhibits a positive immunostimulatoryeffect in the intended immunisation subject without significantdeleterious effects. Specific, non-limiting examples include heat shockproteins from Helicobacter pylori, P. aeruginosa, C. trachomatis and M.leprae, especially the hsp60 group of heat shock proteins.

More particularly, three heat shock proteins are specificallyexemplified herein, namely, M. bovis BCG GroEL-type 65 kDa hsp (hspR65),Recombinant M.tuberculosis DnaK-type 70 kDa hsp (hspR70) and a novelheat shock protein from H.pylori.

The H. pylori heat shock protein (hsp) is a protein whose nucleotide andamino acid sequence is given in FIG. 3 and whose molecular weight is inthe range of 54-62 kDa, preferably about 58-60 kDa. This hsp belongs tothe group of Gram negative bacteria heat shock proteins, hsp60. Ingeneral, hsp are among the most conserved proteins in all livingorganisms, either prokaryotic and eukaroytic, animals and plants, andthe conservation is spread along the whole sequence.

The conjugate may contain one or more heat shock proteins orimmunostimulatory domains thereof. The heat shock proteins may the sameor different. Preferably however, one heat shock protein or a portioncontaining one or more immunostimulatory domains is present.

As used herein, the term “immunostimulatory domain” refers to a regionof a heat shock protein amino acid sequence capable of enhancing theimmune reaction of a subject mammal to a polysaccharide oroligosaccharide component of a conjugate compound including the domain.

An advantage of using only specific domains from complete heat shockproteins is that it is possible selectively not to include domainscommon to human heat shock proteins. For human vaccination this isadvantageous as such regions will not affect the immunostimulatoryeffect of the heat shock protein as they will be recognized as “self”.In addition any immunity that is stimulated against such “self” regionsmight lead to autoimmunity.

Suitable domains of the hsp60 family of heat shock protein areidentified in FIG. 2 by underlining of sequence of reduced homology withthe human heat shock protein. Functional sub domains within the domainsshown in FIG. 2 (SEQ. ID NO: 1); (SEQ. ID NO: 2); (SEQ. ID NO: 3); (SEQ.ID NO: 4); and (SEQ. ID NO: 5) may also be used, as can domain and subdomain combinations.

The skilled man can readily ascertain for a given heat shock proteinwhich domains or epitopes are responsible for the immunostimulatoryaction and prepare modified heat shock protein containing only thosedomains or a sub set thereof.

The oligosaccharide or polysaccharide component of the conjugatecompound may be the complete capsular polysaccharide or oligosaccharideof any pathogenic microorganism against which vaccination is indicatedor a portion thereof capable of eliciting protective immunity. Theoligosaccharide or polysaccharide may be from a single bacteria or fromtwo or more bacteria.

Particular non-limiting examples of bacteria which may be targetedinclude: Haemophilus influenzae Type b (Hib), Streptococcus (includingpneumococcus), Salmonella especially Salmonella typhi, intestinaldisease caused by non-typoidal Salmonella or Shigella bacteria.

According to a particular embodiment of the invention, there have beenprepared conjugates consisting of oligosaccharides of the Meningococci C(MenC) group and hsp.

According to a further particular embodiment of the present inventionthe hsp used for this purpose are hspR65 and hspR70.

In a second aspect of the invention, there is provided a process forproducing conjugate compounds according to the present invention whichcomprises covalently bonding a heat shock protein or portion thereofincluding at least one immunostimulatory domain thereof to at least oneoligosaccharide or polysaccharide.

The oligosaccharide or polysaccharide is preferably isolated from thebacterium to be targetted, but may be produced synthetically.

The heat shock protein may be isolated from its naturally occurringsource or produced synthetically. Preferably the heat shock protein isproduced by recombinant DNA technology using the techniques described inthe general of the description herein.

Preferably the oligosacpharide or polysacoharide is modified prior toconjugation with the heat shock protein or portion thereof to providereactive sites for conjugation. Suitably this involves introducingactive functional groups, such as amino groups at the end groups of theoligosaccharide or polysaccharide. The thus modified oligosaccharide orpolysaccharide may then be activated using a linking group, such assuccinimide and conjugated to the heat shock protein or portion thereof.

In a third aspect of the invention, there is provided a conjugatecompound according to the first aspect of the invention for use as apharmaceutical, preferably as vaccine.

In a fourth aspect of the invention, there is provided the use of theconjugate compound according to the first aspect of the invention in themanufacture of a medicament for vaccination against bacterial infection.

In a fifth aspect of the invention, there is provided a method ofvaccination comprising administering an immunologically effective amountof a conjugate compound according to the first aspect of the invention.

In a sixth aspect of the invention, there is provided a vaccine ortherapeutic composition comprising one or more conjugate compoundsaccording to the first aspect of the invention and a pharmaceuticallyacceptable carrier.

Preferably the composition is a vaccine composition and may includeother excipients such as adjuvants, as necessary (see Section entitled“Vaccines” in the description below).

In a seventh aspect of the invention, there is provided a method for thepreparation of a vaccine comprising bringing one or more conjugatecompounds of the first aspect of the invention into association with apharmaceutically acceptable carrier and optionally an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B shows the results of immunising mice with a hspR65/MenCconjugate and comprises ELISA results for anti-MenC in the blood. InFIG. 1A the mice were preimmunized with BCG and in FIG. 1B they werenot. The Figures show the results with hspR65-MenC (∘) conjugate orhspR70-MenC (⋄) conjugate, in PBS. Control groups of mice were immunisedwith the MenC oligosaccharide alone (Δ) or with a CRM197-MenC conjugatevaccine in aluminium hydroxide (□).

FIGS. 2a-c (SEQ. ID NO: 1); (SEQ. ID NO: 2); (SEQ. ID NO: 3); (SEQ. IDNO: 4); and (SEQ. ID NO: 5) show the amino acid sequence of theHelicobacter pylori heat shock protein and compares it with related heatshock proteins from P. aeruginosa, C.trachomatis, M. leprae and H.sapiens. The bars under the sequence indicate domains on reducedhomology between sequences 1 to 4 and the human heat shock protein.

FIGS. 3a-c (SEQ. ID NO: 1); (SEQ. ID NO: 2); (SEQ. ID NO: 3); (SEQ. IDNO: 4); and (SEQ. ID NO: 5) are the nucleotide and amino acid sequencesof the Helicobacter pylori heat shock protein.

DETAILED DESCRIPTION OF EMBODIMENTS 1. GENERAL METHODOLOGY

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL, SECOND EDITION(1989); DNA CLONING, VOLUMES I AND II (D. N Glover ed. 1985);OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984); NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); TRANSCRIPTION ANDTRANSLATION (B. D. Hames & S. J. Higgins eds. 1984); ANIMAL CELL CULTURE(R. I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press,1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); theseries, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFERVECTORS FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol.155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker,eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY(Academic Press, London), Scopes, (1987), PROTEIN PURIFICATION:PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, N.Y.), andHANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I-IV (D. M. Weir and C. C.Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in thisspecification. All publications, patents, and patent applications citedherein are incorporated by reference.

2. DEFINITIONS

Heat shock proteins that can be used in the present invention includethose polypeptides mentioned above and polypeptides with minor aminoacid variations from the natural amino acid sequence of the protein; inparticular, conservative amino acid replacements are contemplated.

Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids are generally divided into four families: (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3)non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutamine, cystine, serine, threonine, tyrosine. Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. For example, it is reasonably predictable that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid willnot have a major effect on the biological activity. Polypeptidemolecules having substantially the same amino acid sequence as theprotein but possessing minor amino acid substitutions that do notsubstantially affect the functional aspects are within the definition ofthe protein.

A significant advantage of producing the heat shock protein byrecombinant DNA techniques rather than by isolating and purifying aprotein from natural sources is that equivalent quantities of theprotein can be produced by using less starting material than would berequired for isolating the protein from a natural source. Producing theprotein by recombinant techniques also permits the protein to beisolated in the absence of some molecules normally present in cells.Indeed, protein compositions entirely free of any trace of human proteincontaminants can readily be produced because the only human proteinproduced by the recombinant non-human host is the recombinant protein atissue. Potential viral agents from natural sources and viral componentspathogenic to humans are also avoided.

The term “recombinant polynucleotide” as used herein intends apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of a polynucleotide with which it is associated innature, (2) is linked to a polynucleotide other than that to which it islinked in nature, or (3) does not occur in nature.

The term “polynucleotide” as used herein refers to a polymeric form of anucleotide of any length, preferably deoxyribonucleotides, and is usedinterchangeably herein with the terms “oligonucleotide” and “oligomer.”The term refers only to the primary structure of the molecule. Thus,this term includes double- and single-stranded DNA, as well as antisensepolynucleotides. It also includes known types of modifications, forexample, the presence of labels which are known in the art, methylation,end “caps,” substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, replacement with certain types of uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), introduction of pendant moieties, such as, for example, proteins(including nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive species, boron, oxidative moieties,etc.), alkylators (e.g., alpha anomeric nucleic acids, etc.).

By “genomic” is meant a collection or library of DNA molecules which arederived from restriction fragments that have been cloned in vectors.This may include all or part of the genetic material of an organism.

By “cDNA” is meant a complementary mRNA sequence that hybridizes to acomplimentary strand of mRNA.

As used herein, the term “oligomer” refers to both primers and probesand is used interchangeably herein with the term “polynucleotide.” Theterm oligomer does not connote the size of the molecule. However,typically oligomers are no greater than 1000 nucleotides, more typicallyare no greater than 500 nucleotides, even more typically are no greaterthan 250 nucleotides; they may be no greater than 100 nucleotides, andmay be no greater than 75 nucleotides, and also may be no greater than50 nucleotides in length.

The term “primer” as used herein refers to an oligomer which is capableof acting as a point of initiation of synthesis of a polynucleotidestrand when used under appropriate conditions. The primer will becompletely or substantially complementary to a region of thepolynucleotide strand to be copied. Thus, under conditions conducive tohybridization, the primer will anneal to the complementary region of theanalyte strand. Upon addition of suitable reactants, (e.g., apolymerase, nucleotide triphosphates, and the like), the primer will beextended by the polymerizing agent to form a copy of the analyte strand.The primer may be single-stranded or alternatively may be partially orfully double-stranded.

The terms “analyte polynucleotide” and “analyte strand” refer to asingle- or double-stranded nucleic acid molecule which is suspected ofcontaining a target sequence, and which may be present in a biologicalsample.

As used herein, the term “probe” refers to a structure comprised of apolynucleotide which forms a hybrid structure with a target sequence,due to complementarily of at least one sequence in the probe with asequence in the target region. The polynucleotide regions of probes maybe composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.Included within probes are “capture probes” and “label probes”.

As used herein, the term “target region” refers to a region of thenucleic acid which is to be amplified and/or detected. The term “targetsequence” refers to a sequence with which a probe or primer will form astable hybrid under desired conditions.

The term “capture probe” as used herein refers to a polynucleotide probecomprised of a single-stranded polynucleotide coupled to a bindingpartner. The single-stranded polynucleotide is comprised of a targetingpolynucleotide sequence, which is complementary to a target sequence ina target region to be detected in the analyte polynucleotide. Thiscomplementary region is of sufficient length and complementarily to thetarget sequence to afford a duplex of stability which is sufficient toimmobilize the analyte polynucleotide to a solid surface (via thebinding partners). The binding partner is specific for a second bindingpartner; the second binding partner can be bound to the surface of asolid support, or may be linked indirectly via other structures orbinding partners to a solid support.

The term “targeting polynucleotide sequence” as used herein refers to apolynucleotide sequence which is comprised of nucleotides which arecomplementary to a target nucleotide sequence; the sequence is ofsufficient length and complementarily with the target sequence to form aduplex which has sufficient stability for the purpose intended.

The term “binding partner” as used herein refers to a molecule capableof binding a ligand molecule with high specificity, as for example anantigen and an antibody specific therefor. In general, the specificbinding partners must bind with sufficient affinity to immobilize theanalyte copy/complementary strand duplex (in the case of capture probes)under the isolation conditions. Specific binding partners are known inthe art, and include, for example, biotin and avidin or streptavidin,IgG and protein A, the numerous known receptor-ligand couples, andcomplementary polynucleotide strands. In the case of complementarypolynucleotide binding partners, the partners are normally at leastabout 15 bases in length, and may be at least 40 bases in length; inaddition, they have a content of Gs and Cs of at least about 40% and asmuch as about 60%. The polynucleotides may be composed of DNA, RNA, orsynthetic nucleotide analogs.

The term “coupled” as used herein refers to attachment by covalent bondsor by strong non-covalent interactions (e.g., hydrophobic interactions,hydrogen bonds, etc.). Covalent bonds may be, for example, ester, ether,phosphoester, amide, peptide, imide, carbon-sulfur bonds,carbon-phosphorus bonds, and the like.

The term “support” refers to any solid or semi-solid surface to which adesired binding partner may be anchored. Suitable supports includeglass, plastic, metal, polymer gels, and the like, and may take the formof beads, wells, dipsticks, membranes, and the like.

The term “label” as used herein refers to any atom or moiety which canbe used to provide a detectable (preferably quantifiable) signal, andwhich can be attached to a polynucleotide or polypeptide.

As used herein, the term “label probe” refers to a polynucleotide probewhich is comprised of a targeting polynucleotide sequence which iscomplementary to a target sequence to be detected in the analytepolynucleotide. This complementary region is of sufficient length andcomplementarily to the target sequence to afford a duplex comprised ofthe “label probe” and the “target sequence” to be detected by the label.The label probe is coupled to a label either directly, or indirectly viaa set of ligand molecules with high specificity for each other,including multimers.

The term “multimer,” as used herein, refers to linear or branchedpolymers of the same repeating single-stranded polynucleotide unit ordifferent single-stranded polynucleotide units. At least one of theunits has a sequence, length, and composition that permits it tohybridize specifically to a first single-stranded nucleotide sequence ofinterest, typically an analyte or a polynucleotide probe (e.g., a labelprobe) bound to an analyte. In order to achieve such specificity andstability, this unit will normally be at least about 15 nucleotides inlength, typically no more than about 50 nucleotides in length, andpreferably about 30 nucleotides in length; moreover, the content of Gsand Cs will normally be at least about 40%, and at most about 60%. Inaddition to such unit(s), the multimer includes a multiplicity of unitsthat are capable of hybridizing specifically and stably to a secondsingle-stranded nucleotide of interest, typically a labelledpolynucleotide or another multimer. These units are generally about thesame size and composition as the multimers discussed above. When amultimer is designed to be hybridized to another multimer, the first andsecond oligonucleotide units are heterogeneous (different), and do nothybridize with each other under the conditions of the selected assay.Thus, multimers may be label probes, or may be ligands which couple thelabel to the probe.

A “replicon” is any genetic element, e.g., a plasmid, a chromosome, avirus, a cosmid, etc. that behaves as an autonomous unit ofpolynucleotide replication within a cell; i.e., capable of replicationunder its own control. This may include selectable markers.

“PCR” refers to the technique of polymerase chain reaction as describedin Saiki, et al., Nature 324:163 (1986); and Scharf et al., Science(1986) 233:1076-1078; and U.S. Pat. No. 4,683,195; and U.S. Pat. No.4,683,202.

As used herein, x is “heterologous” with respect to y if x is notnaturally associated with y in the identical manner; i.e., x is notassociated with y in nature or x is not associated with y in the samemanner as is found in nature.

“Homology” refers to the degree of similarity between x and y. Thecorrespondence between the sequence from one form to another can bedetermined by techniques known in the art. For example, they can bedetermined by a direct comparison of the sequence information of thepolynucleotide. Alternatively, homology can be determined byhybridization of the polynucleotides under conditions which form stableduplexes between homologous regions (for example, those which would beused prior to S₁ digestion), followed by digestion with single-strandedspecific nuclease(s), followed by size determination of the digestedfragments.

A “vector” is a replicon in which another polynucleotide segment isattached, so as to bring about the replication and/or expression of theattached segment.

“Control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is necessary for expression, and may also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.

An “open reading frame” (ORF) is a region of a polynucleotide sequencewhich encodes a polypeptide; this region may represent a portion of acoding sequence or a total coding sequence.

A “coding sequence” is a polynucleotide sequence which is translatedinto a polypeptide, usually via mRNA, when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a translation start codon at the 5′-terminus and atranslation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to, cDNA, and recombinant polynucleotidesequences.

As used herein, the term “polypeptide” refers to a polymer of aminoacids and does not refer to a specific length of the product; thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide. This term also does not refer to or exclude postexpression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

A polypeptide or amino acid sequence “derived from” a designated nucleicacid sequence refers to a polypeptide having an amino acid sequenceidentical to that of a polypeptide encoded in the sequence, or a portionthereof wherein the portion consists of at least 3-5 amino acids, andmore preferably at least 8-10 amino acids, and even more preferably atleast 11-15 amino acids, or which is immunologically identifiable with apolypeptide encoded in the sequence. This terminology also includes apolypeptide expressed from a designated nucleic acid sequence.

“Immunogenic” refers to the ability of a polypeptide to cause a humonaland/or cellular immune response, whether alone or when linked to acarrier, in the presence or absence of an adjuvant. “Neutralization”refers to an immune response that blocks the infectivity, eitherpartially or fully, of an infectious agent. “Epitope” refers to anantigenic determinant of a peptide, polypeptide, or protein; an epitopecan comprise 3 or more amino acids in a spatial conformation unique tothe epitope. Generally, an epitope consists of at least 5 such aminoacids and, more usually, consists of at least 8-10 such amino acids.Methods of determining spatial conformation of amino acids are known inthe art and include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance. Antibodies that recognize thesame epitope can be identified in a simple immunoassay showing theability of one antibody to block the binding of another antibody to atarget antigen.

“Treatment,” as used herein, refers to prophylaxis and/or therapy (i.e.,the modulation of any disease symptoms). An “individual” indicates ananimal that is susceptible to infection by bacterium possessing anantigenic capsular polysaccharide or oligosaccharide structure andincludes, but is not limited to, primates, including humans. A “vaccine”is an immunogenic, or otherwise capable of eliciting protection againstsuch a bacterium, whether partial or complete, composition useful fortreatment of an individual.

The conjugate compounds of the invention may be used for producingantibodies, either monoclonal or polyclonal, specific to the proteins.The methods for producing these antibodies are known in the art.

“Recombinant host cells”, “host cells,” “cells,” “cell cultures,” andother such terms denote, for example, microorganisms, insect cells, andmammalian cells, that can be, or have been, used as recipients forrecombinant vector or other transfer DNA, and include the progeny of theoriginal cell which has been transformed. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.Examples for mammalian host cells include Chinese hamster ovary (CHO)and monkey kidney (COS) cells.

Specifically, as used herein, “cell line,” refers to a population ofcells capable of continuous or prolonged growth and division in vitro.Often, cell lines are clonal populations derived from a singleprogenitor cell. It is further known in the art that spontaneous orinduced changes can occur in karyotype during storage or transfer ofsuch clonal populations. Therefore, cells derived from the cell linereferred to may not be precisely identical to the ancestral cells orcultures, and the cell line referred to includes such variants. The term“cell lines” also includes immortalized cells. Preferably, cell linesinclude nonhybrid cell lines or hybridomas to only two cell types.

As used herein, the term “microorganism” includes prokaryotic andeukaryotic microbial species such as bacteria and fungi, the latterincluding yeast and filamentous fungi.

“Transformation”, as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for the insertion, for example, direct uptake, transduction,f-mating or electroporation. The exogenous polynucleotide may bemaintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host genome.

By “purified” and “isolated” is meant, when referring to a polypeptideor nucleotide sequence, that the indicated molecule is present in thesubstantial absence of other biological macromolecules of the same type.The term “purified” as used herein preferably means at least 75% byweight, more preferably at least 85% by weight, more preferably still atleast 95% by weight, and most preferably at least 98% by weight, ofbiological macromolecules of the same type present (but water, buffers,and other small molecules, especially molecules having a molecularweight of less than 1000, can be present).

3. EXPRESSION SYSTEMS

Once the appropriate heat shock protein coding sequence is isolated, itcan be expressed in a variety of different expression systems; forexample those used with mammalian cells, baculoviruses, bacteria, andyeast.

3.1. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoteris any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence (e.g.structural gene) into mRNA. A promoter will have a transcriptioninitiating region, which is usually placed proximal to the 5′ end of thecoding sequence, and a TATA box, usually located 25-30 base pairs (bp)upstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element,usually located within 100 to 200 bp upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation, Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed (1989).

Mammalian viral genes are often highly expressed and have a broad hostrange; therefore sequences encoding mammalian viral genes provideparticularly useful promoter sequences. Examples include the SV40 earlypromoter, mouse mammary tumor virus LTR promoter, adenovirus major latepromoter (Ad MLP), and herpes simplex virus promoter. In addition,sequences derived from non-viral genes, such as the murinemetallotheionein gene, also provide useful promoter sequences.Expression may be either constitutive or regulated (inducible),depending on the promoter can be induced with glucocorticoid inhormone-responsive cells.

The presence of an enhancer element (enhancer), combined with thepromoter elements described above, will usually increase expressionlevels. An enhancer is a regulatory DNA sequence that can stimulatetranscription up to 1000-fold when linked to homologous or heterologouspromoters, with synthesis beginning at the normal RNA start site.Enhancers are also active when they are placed upstream or downstreamfrom the transcription initiation site, in either normal or flippedorientation, or at a distance of more than 1000 nucleotides from thepromoter, Maniatis et al., Science 236:1237 (1989); Alberts et al.Molecular Biology of the Cell, 2nd ed (1989). Enhancer elements derivedfrom viruses may be particularly useful, because they usually have abroader host range. Examples include the SV40 early gene enhancer,Dijkema et al (1985) EMBO J. 4:761, and the enhancer/promoters derivedfrom the long terminal repeat (LTR) of the Rous Sarcoma Virus, Gorman etal. (1982) Proc. Natl. Acad. Sci. 79:6777, and from humancytomegalovirus, Boshart et al. (1985) Cell 41:5221. Additionally, someenhancers are regulatable and become active only in the presence of aninducer, such as a hormone or metal ion, Sassone-Corsi et al. (1986)Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237.

A DNA molecule may be expressed intracellularly in mammalian cells. Apromoter sequence may be directly linked with the DNA molecule, in whichcase the first amino acid at the N-terminus of the recombinant proteinwill always be a methionine, which is encoded by the ATG start codon. Ifdesired, the N-terminus may be cleaved from the protein by in vitroincubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provides forsecretion of the foreign protein in mammalian cells. Preferably, thereare processing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell. Theadenovirus tripartite leader is an example of a leader sequence thatprovides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-transcriptional cleavage and polyadenylation,Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988)“Termination and 3′ end processing of eukaryotic RNA. In Transcriptionand splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) TrendsBiochem. Sci. 14:105. These sequences direct the transcription of anmRNA which can be translated into the polypeptide encoded by the DNA.Examples of transcription terminator/polyadenylation signals includethose derived from SV40, Sambrook et al (1989), Molecular Cloning: ALaboratory Manual.

Some genes may be expressed more efficiently when introns (also calledintervening sequences) are present. Several cDNAs, however, have beenefficiently expressed from vectors that lack splicing signals (alsocalled splice donor and acceptor sites), see e.g., Gething and Sambrook(1981) Nature 293:620. Introns are intervening noncoding sequenceswithin a coding sequence that contain splice donor and acceptor sites.They are removed by a process called “splicing,” followingpolyadenylation of the primary transcript, Nevins (1983) Annu. Rev.Biochem. 52:441; Green (1986) Annu. Rev. Genet. 20:671; Padgett et al.(1986) Annu. Rev. Biochem. 55:1119; Krainer and Maniatis (1988) “RNAsplicing,” In Transcription and splicing (ed. B. D. Hames and D. M.Glover).

Usually, the above-described components, comprising a promoter,polyadenylation signal, and transcription termination sequence are puttogether into expression constructs. Enhancers, introns with functionalsplice donor and acceptor sites, and leader sequences may also beincluded in an expression construct, if desired. Expression constructsare often maintained in a replicon, such as an extrachromosomal element(e.g., plasmids) capable of stable maintenance in a host, such asmammalian cells or bacteria. Mammalian replication systems include thosederived from animal viruses, which require transacting factors toreplicate. For example, plasmids containing the replication systems ofpapovaviruses, such as SV40, Gluzman (1981) Cell 23:175, orpolyomavirus, replicate to extremely high copy number in the presence ofthe appropriate viral T antigen. Additional examples of mammalianreplicons include those derived from bovine papillomavirus andEpstein-Barr virus. Additionally, the replicon may have two replicationsystems, thus allowing it to be maintained, for example, in mammaliancells for expression and in a procaryotic host for cloning andamplification. Examples of such mammalian-bacteria shuttle vectorsinclude pMT2, Kaufman et al. (1989) Mol. Cell. Biol. 9:946, and PHEBO,Shimizu et al. (1986) Mol. Cell. Biol. 6:1074.

The transformation procedure used depends upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to, Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g.,Hep G2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into asuitable insect expression vector, and is operably linked to the controlelements within that vector. Vector construction employs techniqueswhich are known in the art.

Generally, the components of the expression system include a transfervector, usually a bacterial plasmid, which contains both a fragment ofthe baculovirus genome, and a convenient restriction site for insertionof the heterologous gene or genes to be expressed; a wild typebaculovirus with a sequence homologous to the baculovirus-specificfragment in the transfer vector (this allows for the homologousrecombination of the heterologous gene in to the baculovirus genome);and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfervector, the vector and the wild type viral genome are transfected intoan insect host cell where the vector and viral genome are allowed torecombine. The packaged, recombinant virus is expressed and recombinantplaques are identified and purified. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego, Calif. (“MAXBAC™” kit(expression system)). These techniques are generally known to thoseskilled in the art and fully described in Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter“Summers and Smith”).

Prior to inserting the DNA sequence encoding the protein into thebaculovirus genome, the above-described components, comprising apromoter, leader (if desired), coding sequence of interest, andtranscription termination sequence, are usually assembled into anintermediate transplacement construct (transfer vector). This constructmay contain a single gene and operably linked regulatory elements;multiple genes, each with its owned set of operably linked regulatoryelements; or multiple genes, regulated by the same set of regulatoryelements. Intermediate transplacement constructs are often maintained ina replicon, such as an extrachromosomal element (e.g., plasmids) capableof stable maintenance in a host, such as a bacterium. The replicon willhave a replication system, thus allowing it to be maintained in asuitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedron polyadenylation signal(Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a procaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (e.g. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region usually includes an RNA polymerase binding site and atranscription initiation site. A baculovirus transfer vector may alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in a viralinfection cycle, provide particularly useful promoter sequences.Examples include sequences derived from the gene encoding the viralpolyhedron protein, Friesen et al., (1986) “The Regulation ofBaculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses(ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the geneencoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes forsecreted insect or baculovirus proteins, such as the baculoviruspolyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively,since the signals for mammalian cell posttranslational modifications(such as signal peptide cleavage, proteolytic cleavage, andphosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells,leaders of non-insect origin, such as those derived from genes encodinghuman α-interferon, Maeda et al., (1985), Nature 315:592; humangastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell.Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci.USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; andhuman glucocerebrosidase, Martin et al. (1988) DNA 7:99, can also beused to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressedintracellularly or, if it is expressed with the proper regulatorysequences, it can be secreted. Good intracellular expression of nonfusedforeign proteins usually requires heterologous genes that ideally have ashort leader sequence containing suitable translation initiation signalspreceding an ATG start signal. If desired, methionine at the N-terminusmay be cleaved from the mature protein by in vitro incubation withcyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are notnaturally secreted can be secreted from the insect cell by creatingchimeric DNA molecules that encode a fusion protein comprised of aleader sequence fragment that provides for secretion of the foreignprotein in insects. The leader sequence fragment usually encodes asignal peptide comprised of hydrophobic amino acids which direct thetranslocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding theexpression product precursor of the protein, an insect cell host isco-transformed with the heterologous DNA of the transfer vector and thegenomic DNA of wild type baculovirus—usually by co-transfection. Thepromoter and transcription termination sequence of the construct willusually comprise a 2-5 kb section of the baculovirus genome. Methods forintroducing heterologous DNA into the desired site in the baculovirusvirus are known in the art. (See Summers and Smith; Ju et al. (1987);Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers(1989)). For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. Miller et al., (1989), Bioessays 4:91.

The DNA sequence, when cloned in place of the polyhedrin gene in theexpression vector, is flanked both 5′ and 3′ by polyhedrin-specificsequences and is positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packagedinto an infectious recombinant baculovirus. Homologous recombinationoccurs at low frequency (between about 1% and about 5%); thus, themajority of the virus produced after cotransfection is still wild-typevirus. Therefore, a method is necessary to identify recombinant viruses.An advantage of the expression system is a visual screen allowingrecombinant viruses to be distinguished. The polyhedrin protein, whichis produced by the native virus, is produced at very high levels in thenuclei of infected cells at late times after viral infection.Accumulated polyhedrin protein forms occlusion bodies that also containembedded particles. These occlusion bodies, up to 15 μm in size, arehighly refractile, giving them a bright shiny appearance that is readilyvisualized under the light microscope. Cells infected with recombinantviruses lack occlusion bodies. To distinguish recombinant virus fromwild-type virus, the transfection supernatant is plaqued onto amonolayer of insect cells by techniques known to those skilled in theart. Namely, the plaques are screened under the light microscope for thepresence (indicative of wild-type virus) or absence (indicative ofrecombinant virus) of occlusion bodies. “Current Protocols inMicrobiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990);Summers and Smith; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia: Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (PCT Pub. No. Wo 89/046699; Carbonell etal., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith etal., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al.(1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both directand fusion expression of heterologous polypeptides in abaculovirus/expression system; cell culture technology is generallyknown to those skilled in the art. See, e.g., Summers and Smith.

The modified insect cells may then be grown in an appropriate nutrientmedium, which allows for stable maintenance of the plasmid(s) present inthe modified insect host. Where the expression product gene is underinducible control, the host may be grown to high density, and expressioninduced. Alternatively, where expression is constitutive, the productwill be continuously expressed into the medium and the nutrient mediummust be continuously circulated, while removing the product of interestand augmenting depleted nutrients. The product may be purified by suchtechniques as chromatography, e.g., HPLC, affinity chromatography, ionexchange chromatography, etc.; electrophoresis; density gradientcentrifugation; solvent extraction, or the like. As appropriate, theproduct may be further purified, as required, so as to removesubstantially any insect proteins which are also secreted in the mediumor result from lysis of insect cells, so as to provide a product whichis at least substantially free of host debris, e.g., proteins, lipidsand polysaccharides.

In order to obtain protein expression, recombinant host cells derivedfrom the transformants are incubated under conditions which allowexpression of the recombinant protein encoding sequence. Theseconditions will vary, dependent upon the host cell selected. However,the conditions are readily ascertainable to those of ordinary skill inthe art, based upon what is known in the art.

iii. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterialpromoter is any DNA sequence capable of binding bacterial RNA polymeraseand initiating the downstream (3″) transcription of a coding sequence(e.g. structural gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region usually includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter may also have a second domain called an operator,that may overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein may bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression may occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation may be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in E. coli, Raibaudet al. (1984) Annu. Rev. Genet. 18:173. Regulated expression maytherefore be either positive or negative, thereby either enhancing orreducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac), Chang etal. (1977) Nature 198:1056, and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp), Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al.(1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPO Publ. Nos.036 776 and 121 775. The g-laotamase (bla) promoter system, Weissmann(1981) “The cloning of interferon and other mistakes.” In Interferon 3(ed. I. Gresser), bacteriophage lambda PL, Shimatake et al. (1981)Nature 292:128, and T5, U.S. Pat. No. 4,689,406, promoter systems alsoprovide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter, U.S. Pat. No. 4,551,433. Forexample, the tac promoter is a hybrid trp-lac promoter comprised of bothtrp promoter and lac operon sequences that is regulated by the lacrepressor, Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc.Natl. Acad. Sci. 80:21. Furthermore, a bacterial promoter can includenaturally occurring promoters of non-bacterial origin that have theability to bind bacterial RNA polymerase and initiate transcription. Anaturally occurring promoter of non-bacterial origin can also be coupledwith a compatible RNA polymerase to produce high levels of expression ofsome genes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system, Studier et al. (1986)J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci.82:1074. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EPO Publ. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon, Shine et al. (1975) Nature 254:34. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA, Steitz et al. (1979) “Genetic signals and nucleotide sequences inmessenger RNA.” In Biological Regulation and Development: GeneExpression (ed. R. F. Goldberger). To express eukaryotic genes andprokaryotic genes with weak ribosome-binding site, Sambrook et al.(1989), Molecular Cloning: A Laboratory Manual.

A DNA molecule may be expressed intracellularly. A promoter sequence maybe directly linked with the DNA molecule, in which case the first aminoacid at the N-terminus will always be a methionine, which is encoded bythe ATG start codon. If desired, methionine at the N-terminus may becleaved from the protein by in vitro incubation with cyanogen bromide orby either in vivo on in vitro incubation with a bacterial methionineN-terminal peptidase (EPO Publ. No. 219 237).

Fusion proteins provide an alternative to direct expression. Usually, aDNA sequence encoding the N-terminal portion of an endogenous bacterialprotein, or other stable protein, is fused to the 5′ end of heterologouscoding sequences. Upon expression, this construct will provide a fusionof the two amino acid sequences. For example, the bacteriophage lambdacell gene can be linked at the 5′ terminus of a foreign gene andexpressed in bacteria. The resulting fusion protein preferably retains asite for a processing enzyme (factor Xa) to cleave the bacteriophageprotein from the foreign gene, Nagai et al. (1984) Nature 309:810.Fusion proteins can also be made with sequences from the lacZ, Jia etal. (1987) Gene 60:197, trpE, Allen et al. (1987) J. Biotechnol. 5:93;Makoff et al. (1989) J. Gen. Microbiol. 135:11, and EPO Publ. No. 324647, genes. The DNA sequence at the junction of the two amino acidsequences may or may not encode a cleavable site. Another example is aubiquitin fusion protein. Such a fusion protein is made with theubiquitin region that preferably retains a site for a processing enzyme(e.g. ubiquitin specific processing-protease) to cleave the ubiquitinfrom the foreign protein. Through this method, native foreign proteincan be isolated. Miller et al. (1989) Bio/Technology 7:698.

Alternatively, foreign proteins can also be secreted from the cell bycreating chimeric DNA molecules that encode a fusion protein comprisedof a signal peptide sequence fragment that provides for secretion of theforeign protein in bacteria, U.S. Pat. No. 4,336,336. The signalsequence fragment usually encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).Preferably there are processing sites, which can be cleaved either invivo or in vitro encoded between the signal peptide fragment and theforeign gene.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA). Masui et al. (1983), in: Experimental Manipulation of GeneExpression; Ghrayeb et al. (1984) EMBO J. 3:2437 and the E. colialkaline phosphatase signal sequence (phoA), Oka et al. (1985) Proc.Natl. Acad. Sci. 82:7212. As an additional example, the signal sequenceof the alpha-amylase gene from various Bacillus strains can be used tosecrete heterologous proteins from B. subtilis. Palva et al. (1982)Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. No. 244 042.

Usually, transcription termination sequences recognized by bacteria areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Transcription termination sequencesfrequently include DNA sequences of about 50 nucleotides capable offorming stem loop structures that aid in terminating transcription.Examples include transcription termination sequences derived from geneswith strong promoters, such as the trp gene in E. coli as well as otherbiosynthetic genes.

Usually, the above-described components, comprising a promoter, signalsequence (if desired), coding sequence of interest, and transcriptiontermination sequence, are put together into expression constructs.Expression constructs are often maintained in a replicon, such as anextrachromosomal element (e.g., plasmids) capable of stable maintenancein a host, such as bacteria. The replicon will have a replicationsystem, thus allowing it to be maintained in a procaryotic host eitherfor expression or for cloning and amplification. In addition, a repliconmay be either a high or low copy number plasmid. A high copy numberplasmid will generally have a copy number ranging from about 5 to about200, and usually about 10 to about 150. A host containing a high copynumber plasmid will preferably contain at least about 10, and morepreferably at least about 20 plasmids. Either a high or low copy numbervector may be selected, depending upon the effect of the vector and theforeign protein on the host.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to the bacterial chromosomethat allows the vector to integrate. Integrations appear to result fromrecombinations between homologous DNA in the vector and the bacterialchromosome. For example, integrating vectors constructed with DNA fromvarious Bacillus strains integrate into the Bacillus chromosome (EPOPubl. No. 127 328). Integrating vectors may also be comprised ofbacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and may include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline. Davies et al. (1978) Annu.Rev.Microbiol. 32:469. Selectable markers may also include biosyntheticgenes, such as those in the histidine, tryptophan, and leucinebiosynthetic pathways.

Alternatively, some of the above-described components can be puttogether in transformation vectors. Transformation vectors are usuallycomprised of a selectable marker that is either maintained in a repliconor developed into an integrating vector.

Expression and transformation vectors, either extra-chromosomalreplicons or integrating vectors, have been developed for transformationinto many bacteria. For example, expression vectors have been developedfor, inter alia, the following bacteria: Bacillus subtilis, Palv et al.(1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259 and063 953; PCT Publ. No. WO 84/04541; E. coli, Shimatake et al. (1981)Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986)J. Mol. Biol. 189:113; EPO Publ. Nos. 036 776, 136 829 and 136 907;Streptococcus cremoris, Powell et al. (1988) Appl. Environ. Microbiol.54:655; Streptococcus lividans, Powell et al. (1988) Appl. Environ.Microbiol. 54:655; and Streptomyces lividans, U.S. Pat. No. 4,745,056.

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and usually include either the transformation of bacteriatreated with CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Transformation procedures usually vary with the bacterial species to betransformed. See, e.g., Masson et al. (1989) FEMS Microbiol. Lett.60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPOPubl. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541, for Bacillus;Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990)J. Bacteriol. 172:949, for Campylobacter; Cohen et al. (1973) Proc.Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res.16:6127; Kushner (1978) “An improved method for transformation of E.coli with ColE1-derived plasmids,” In Genetic Engineering: Proceedingsof the International Symposium on Genetic Engineering (eds. H. W. Boyerand S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo(1988) Biochim. Biophys. Acta 949:318, for Escherichia; Chassy et al.(1987) FEMS Microbiol. Lett. 44:173, for Lactobacillus; Fiedler et al.(1988) Anal. Biochem 170:38, for Pseudomonas; Augustin et al. (1990)FEMS Microbiol. Lett. 66:203, for Staphylococcus; Barany et al. (1980)J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcuslactis by electroporation, in: Streptococcal Genetics (ed. J. Ferrettiand R. Curtiss III); Perry et al. (1981) Infec. Immun. 32:1295; Powellet al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987)Proc. 4th Evr. Cong. Biotechnology 1:412, for Streptococcus.

iv. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in theart. A yeast promoter is any DNA sequence capable of binding yeast RNApolymerase and initiating the downstream (3′) transcription of a codingsequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regionusually includes an RNA polymerase binding site (the “TATA Box”) and atranscription initiation site. A yeast promoter may also have a seconddomain called an upstream activator sequence (UAS), which, if present,is usually distal to the structural gene. The UAS permits regulated(inducible) expression. Constitutive expression occurs in the absence ofa UAS. Regulated expression may be either positive or negative, therebyeither enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway,therefore sequences encoding enzymes in the metabolic pathway provideparticularly useful promoter sequences. Examples include alcoholdehydrogenase (ADH) (EPO Publ. No. 284 044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK) (EPO Publ. No. 329 203). The yeastPHO5 gene, encoding acid phosphatase, also provides useful promotersequences, Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1.

In addition, synthetic promoters which do not occur in nature alsofunction as yeast promoters. For example, UAS sequences of one yeastpromoter may be joined with the transcription activation region ofanother yeast promoter, creating a synthetic hybrid promoter. Examplesof such hybrid promoters include the ADH regulatory sequence linked tothe GAP transcription activation region (U.S. Pat. No. 4,876,197 andU.S. Pat. No. 4,880,734). Other examples of hybrid promoters includepromoters which consist of the regulatory sequences of either the ADH2,GAL4, GAL10, or PH05 genes, combined with the transcriptional activationregion of a glycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164556). Furthermore, a yeast promoter can include naturally occurringpromoters of non-yeast origin that have the ability to bind yeast RNApolymerase and initiate transcription. Examples of such promotersinclude, inter alia, Cohen et al. (1980) Proc. Natl. Acad. Sci. USA77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981)Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979) “TheExpression of Bacterial Antibiotic Resistance Genes in the YeastSaccharomyces cerevisiae,” in: Plasmids of Medical, Environmental andCommercial Importance (eds. K. N. Timmis and A. Puhler);Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980)Curr. Genet. 2:109.

A DNA molecule may be expressed intracellularly in yeast. A promotersequence may be directly linked with the DNA molecule, in which case thefirst amino acid at the N-terminus of the recombinant protein willalways be a methionine, which is encoded by the ATG start codon. Ifdesired, methionine at the N-terminus may be cleaved from the protein byin vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, aswell as in mammalian, baculovirus, and bacterial expression systems.Usually, a DNA sequence encoding the N-terminal portion of an endogenousyeast protein, or other stable protein, is fused to the 5′ end ofheterologous coding sequences. Upon expression, this construct willprovide a fusion of the two amino acid sequences. For example, the yeastor human superoxide dismutase (SOD) gene, can be linked at the 5′terminus of a foreign gene and expressed in yeast. The DNA sequence atthe junction of the two amino acid sequences may or may not encode acleavable site. See e.g., EPO Publ. No. 196 056. Another example is aubiquitin fusion protein. Such a fusion protein is made with theubiquitin region that preferably retains a site for a processing enzyme(e.g. ubiquitin-specific processing protease) to cleave the ubiquitinfrom the foreign protein. Through this method, therefore, native foreignprotein can be isolated (see, e.g., PCT Publ. No. WO 88/024066).

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provide forsecretion in yeast of the foreign protein. Preferably, there areprocessing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase gene (EPO Publ. No.012 873; JPO Publ. No. 62,096,086) and the A-factor gene (U.S. Pat. No.4,588,684). Alternatively, leaders of non-yeast origin, such as aninterferon leader, exist that also provide for secretion in yeast (EPOPubl. No. 060 057).

A preferred class of secretion leaders are those that employ a fragmentof the yeast alpha-factor gene, which contains both a “pre” signalsequence, and a “pro” region. The types of alpha-factor fragments thatcan be employed include the full-length pre-pro alpha factor leader(about 83 amino acid residues) as well as truncated alpha-factor leaders(usually about 25 to about 50 amino acid residues) (U.S. Pat. No.4,546,083 and U.S. Pat. No. 4,870,008; EPO Publ. No. 324 274).Additional leaders employing an alpha-factor leader fragment thatprovides for secretion include hybrid alpha-factor leaders made with apresequence of a first yeast, but a pro-region from a second yeastalphafactor. (See e.g., PCT Publ. No. WO 89/02463.)

Usually, transcription termination sequences recognized by yeast areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Examples of transcription terminatorsequence and other yeast-recognized termination sequences, such as thosecoding for glycolytic enzymes.

Usually, the above-described components, comprising a promoter, leader(if desired), coding sequence of interest, and transcription terminationsequence, are put together into expression constructs. Expressionconstructs are often maintained in a replicon, such as anextrachromosomal element (e.g., plasmids) capable of stable maintenancein a host, such as yeast or bacteria. The replicon may have tworeplication systems, thus allowing it to be maintained, for example, inyeast for expression and in a procaryotic host for cloning andamplification. Examples of such yeast-bacteria shuttle vectors includeYEp24, Botstein et al. (1979) Gene 8:17-24; pCl/1, Brake et al. (1984)Proc. Natl. Acad. Sci USA 81:4642-4646; and YRp17, Stinchcomb et al.(1982) J. Mol. Biol. 158:157. In addition, a replicon may be either ahigh or low copy number plasmid. A high copy number plasmid willgenerally have a copy number ranging from about 5 to about 200, andusually about 10 to about 150. A host containing a high copy numberplasmid will preferably have at least about 10, and more preferably atleast about 20. A high or low copy number vector may be selected,depending upon the effect of the vector and the foreign protein on thehost.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome, Orr-Weaver et al. (1983) Methods in Enzymol.101:228-245. An integrating vector may be directed to a specific locusin yeast by selecting the appropriate homologous sequence for inclusionin the vector. One or more expression construct may integrate, possiblyaffecting levels of recombinant protein produced, Rine et al. (1983)Proc. Natl. Acad. Sci. USA 80:6750. The chromosomal sequences includedin the vector can occur either as a single segment in the vector, whichresults in the integration of the entire vector, or two segmentshomologous to adjacent segments in the chromosome and flanking theexpression construct in the vector, which can result in the stableintegration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers may include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP1, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker may also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP1 allows yeast to grow in the presence of copper ions.Butt et al. (1987) Microbiol, Rev. 51:351.

Alternatively, some of the above-described components can be puttogether into transformation vectors. Transformation vectors are usuallycomprised of a selectable marker that is either maintained in a repliconor developed into an integrating vector.

Expression and transformation vectors, either extrachromosomal repliconsor integrating vectors, have been developed for transformation into manyyeasts. For example, expression vectors have been developed for, interalia, the following yeasts: Candida albicans, Kurtz, et al. (1986) Mol.Cell. Biol. 6:142; Candida maltosa, Kunze, et al. (1985) J. BasicMicrobiol. 25:141; Hansenula polymorpha, Gleeson, et al. (1986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302;Kluyveromyces fragilis, Das, et al. (1984) J. Bacteriol. 158:1165;Kluyveromyces lactis, De Louvencourt et al. (1983) J. Bacteriol.154:737; Van den Berg et al. (1990) Bio/Technology 8:135; Pichiaquillerimondii, Kunze et al. (1985) J. Basic Microbiol. 25:141; Pichiapastoris, Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. No.4,837,148 and U.S. Pat. No. 4,929,555; Saccharomyces cerevisiae, Hinnenet al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J.Bacteriol. 153:163; Schizosaccharomyces pombe, Beach et al. (1981)Nature 300:706; and Yarrowia lipolytica, Davidow, et al. (1985) Curr.Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49.

Methods of introducing exogenous DNA into yeast hosts are well-known inthe art, and usually include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed. Seee.g., Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J.Basic Microbiol. 25:141, for Candida; Gleeson et al. (1986) J. Gen.Microbioy. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302,for Hansenula; Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourtet al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990)Bio/Technology 8:135, for Kluvveromyces; Cregg et al. (1985) Mol. Cell.Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat.No. 4,837,148 and U.S. Pat. No. 4,929,555, for Pichia; Hinnen et al.(1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al. (1983) J.Bacteriol. 153:163, for Saccharomyces; Beach et al. (1981) Nature300:706, for Schizosaccharomyces; Davidow et al. (1985) Curr. Genet.10:39; Gaillardin et al. (1985) Curr. Genet. 10:49, for Yarrowia.

4. VACCINES

Each of the conjugate compounds discussed herein may be used as a solevaccine candidate or in combination with one or more other antigens fromother pathogenic sources. These vaccines may either be prophylactic (toprevent infection) or therapeutic (to treat disease after infection).

Such vaccines comprise the conjugate compound usually in combinationwith “pharmaceutically acceptable carriers”, which include any carrierthat does not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. Additionally, these carriers mayfunction as immunostimulating agents (“adjuvants”). Furthermore, theantigen may be conjugated to a bacterial toxoid, such as a toxoid fromdiphtheria, tetanus etc.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: (1) aluminum salts (alum), such as aluminumhydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-wateremulsion formulations (with or without other specific immunostimulatingagents such as muramyl peptides (see below) or bacterial cell wallcomponents), such as for example (a) MF59 (PCT Publ. No. WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE (see below), although not required)formulated into submicron particles using a microfluidizer such as Model110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP (see below) either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) RIBI™(adjuvant system) (RAS), (Ribi Immunochem, Hamilton, Mont.) containing2% Squalene, 0.2% Tween 80, and one or more bacterial cell wallcomponents from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS DETOX™ (monophosphorlipid A+cell wall skeleton); (3) saponinadjuvants, such as STIMULON™ (saponin adjuvant) (Cambridge Bioscience,Worcester, Mass.) may be used or particles generated therefrom such asISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant(CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such asinterleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor(M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances thatact as immunostimulating agents to enhance the effectiveness of thecomposition. Alum and MF59 are preferred.

As mentioned above, muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

The immunogenic compositions (e.g., the antigen, pharmaceuticallyacceptable carrier, and adjuvant) typically will contain diluents, suchas water, saline, glycerol, ethanol, etc. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect, as discussed above underpharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the antigenic polypeptides, as well as any other ofthe above-mentioned components, as needed. By “immunologically effectiveamount”, it is meant that the administration of that amount to anindividual, either in a single dose or as part of a series, is effectivefor treatment or prevention. This amount varies depending upon thehealth and physical condition of the individual to be treated, thetaxonomic group of individual to be treated (e.g., nonhuman primate,primate, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials.

The immunogenic compositions are conventionally administeredparenterally, e.g., by injection, either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule. The vaccine may be administered inconjunction with other immunoregulatory agents.

5. EXAMPLE 1

Conjugate compounds comprising polysaccharides of the Meningococci Cgroup and heat shock proteins hspR70 and hspR65 were constructed andtested for vaccine efficacy

5.1. Purification of Polysaccharides of the Meningococci C (MenC) Group,and Production of MenC Oligosaccharides

The meningococcal polysaccharide of group C was purified as described inFrasc C. E. “Advances in Biotechnological Processes: Bacterial vaccines”(A. Mizrahi, ed.), vol. 13, pp. 123-145, Wiley-Liss Inc., New York(1990). The purified polysaccharide (10 mg/ml) was depolymerised byhydrolysis in 0.01 M acetate buffer of pH 5, at 100° C. for 8 hours. Theresulting product was analysed by analytical chromatography (on Sephadex6-50) and exhibited a Kd (distribution coefficient) of 0.27.

5.2. Introduction of a Primary Amine Group into the Terminal Groups ofthe Oligosaccharide

0.5 M of ammonium chloride and 0.15 M of sodium cyanoborohydride wereadded to the solution obtained from the hydrolysis. The pH was raised to7 and the resulting solution was kept at 35° C. for one week. Theoligosaccharide was then purified by chromatography on Sephadex 6-15),the void volume fractions containing chemical activity in respect of theamine groups and carbohydrate groups being collected while thosecontaining monomeric sugars and excess of reagents were discarded. Theresulting MenC oligosaccharide was characterised by determining theamine groups, the sialic acid groups and the O-acetyl group. Thefollowing molar ratios were obtained: sialic acid/amine groups=20,O-acetyl/sialic acid=0.84.

5.3. Preparation of Heat Shock Proteins

M. bovis BCG GroEL-type 65 kDa hsp (hspR65) was expressed from arecombinant E.coli K12 strain harbouring plasmid pRIB1300 (Thole et al,Infect. Immun. 1985, 50, 800:Van Eden et al, Nature, 1988, 331,171) andpurified as described in Thole et al, Infect. Immun., 1987, 55,1466.

Recombinant M.tuberculosis DnaK-type 70 kDa hsp (hspR70) was obtainedand purified by ATP-agarose chromatography (Mehlert et al Mol.Microbiol., 1989, 3,125).

5.4. Preparation of the Glycoconjugates of MenC Polysaccharide andhspR65 and hspR70

The MenC amino-oligosaccharide was dissolved in dimethylsulphoxide with10% of H₂O and then caused to react with a 12-fold excess (relative tothe amine groups) of the N-hydroxysuccinimide ester of adipic acid[prepared according to Hill et al. “FEBS LETT.” 102:282 (1979)]. Afterpurification by precipitation with dioxane (1-4-fold amount) theactivated oligosaccharide was dried in vacuo and analysed for itscontent of N-hydroxysuccinimino ester. HspR65 and hspR70 in an amount of5 mg/ml of 0.1 M phosphate buffer of pH 7 were then caused to react witha 300-fold molar excess of activated oligosaccharide. Theglycoconjugates respectively obtained were freed from the unreactedoligosaccharides by chromatography, filtered and stored at 4° C. Theratio of their content of sialic acid to the percentage of sialic acidin the MenC polysaccharide starting material represents the amount ofoligosaccharide which has been coupled. The protein content of thepreparation was confirmed by the method of Lowry “J. Biol. Chem.”193:265 (1951). In particular, the conjugate with hspR70 has a proteincontent of 310 μg/ml and a saccharide content of 76 μg/ml whilst theconjugate with hspR65 has a protein content of 180 μg/ml and asaccharide content of 97 μg/ml.

5.5. Mice and Immunisation BALB/c (H-2^(d)), C567BL/6 (H-2^(b)) andCBA/J (H-2^(k)) female mice, 8-12 weeks old, were bred from in ourbreeding facilities.

The starting couples were provided by Jackson Laboratory, Bar Harbor,ME. BALB/c nu/nu athymic mice were obtained from Iffa Credo, L'Arbresle,France.

On day 0, each mouse received intraperitoneally 10⁶ CFU of BCG (or PBSin the case of the control group), followed by 2 doses of conjugate ondays 14 and 35 (in PBS).

Control groups received the MenC oligosaccharide alone, or the MenColigosaccharide—CRM197 conjugate vaccine adsorbed on aluminium hydroxide(1 mg/dose, in 0.5 ml). In each immunisation, the mice received 2 μg ofMenC oligosaccharide, which corresponded to 8.7 μg of MenColigosaccharide—CRM197 conjugate vaccine, 8.4 μg of the MenColigosaccharide—hspR70 conjugate or 3.7 μg of the MenColigosaccharide—hspR65 conjugate.

5.6. Determination of the Antibodies According to the ELISA Method

Each week, blood was taken from the retro-orbital plexus of the mouseand the antibodies were titrated with ELISA.

For determination of IgG anti-MenC antibodies, flat-based plates with 96wells were covered with MenC polysaccharide (5 μg/ml) (Nunc ImmunoplateI, Nunc, Roskilde, Denmark) in PBS, pH 7.4, by overnight incubation at37° C. After repeated washings with PBS containing 0.05% of Tween-20(PBS-T) and incubation for one hour at 37° C. with 200 μl of PBS-Tcontaining 5% of FCS, the wells were incubated overnight at 4° C. with100 μl of mouse serum diluted in PBS-T containing 5% of FCS. Afterrepeated washings, the plates were again incubated for 3 hours at 37° C.with 100 μl of an appropriate dilution of an IgG anti-mouse anti-serumconjugated to peroxidase.

The presence of specific antibodies was revealed by addition of2,2′-azino-bis-(3-ethylbenzothiazoline-sulphonic acid) (ABTS; Kirkegaardand Pery Laboratories Inc., Gaithersburg, Md.) as the substrate. Theresults were measured in terms of the absorbance at 414 nm. Samples ofserum with an absorbance of less than 0.2 at the first dilution tested(1:50) were considered negative.

5.7. Carrier Effect of hpsR65 and hpsR70 Conjugated to Oligosaccharide

HspR65 and hspR70-MenC oligosaccharide conjugates were used to immuniseBALB/c and C57BL/6 mice which had previously been sensitised with BCG orwere non-sensitised.

As a control, groups of mice received MenC only or CRM 197-MenCconjugate vaccine in aluminium hydroxide.

FIG. 1 shows that anti-MenC polysaccharide IgG anti-bodies were producedafter immunisation with hsp-MenC conjugates in amounts comparable to, orgreater than (C57BL/6 in FIG. 1A), those measured in the case of a CRM197-MenC conjugate vaccine in aluminium hydroxide. This effect wasobserved not only in the case of the absence of adjuvant but also, inthe case of the hspR70-MenC conjugate, in the absence of sensitisationwith BCG (FIG. 1B). These results make it possible to state thatimmunisation using mycobacterial hsp in the absence of adjuvants and ofsensitisation with BCG can indeed be achieved also with oligosaccharideantigens.

Accordingly it has been confirmed that the molecules of hsp, inparticular hspR70, exert a powerful carrier effect also in mice notsensitised with BCG, and that hsp acts as a potent carrier of moleculescapable of inducing IgG antibodies which are specific against thepolysaccharide transported, even in the absence of the adjuvants.

This potent carrier effect of the mycobacterial hsp, exerted in theabsence of adjuvants and of pre-sensitisation, makes the conjugatesdescribed particularly useful for the development of novel vaccinesagainst bacterial infections.

6. EXAMPLE 2

A novel H.pylori heat shock protein was identified and produced usingrecombinant DNA techniques.

6.1. Materials and Methods

6.1.1. H. pylori strains and growth conditions

H. pylori strains used were: CCUG 17874, G39 and G33 (isolated fromgastric biopsies in the hospital of Grosseto, Italy), Pylo 2U+and Pylo2U− (provided by F. Megraud, hospital Pellegzin, Bordeaux, France), BA96(isolated by gastric biopsies at the University of Siena, Italy). StrainPylo 2U+is noncytotoxic; strain Pylo 2U− is noncytotoxic andurease-negative. All strains were routinely grown on Columbia agarcontaining 0.2% of cyclodextrin, 5 μg/ml of cefsulodin and 5 μg/ml ofamphotericin B under microaerophilic conditions for 5-6 days at 37° C.Cells were harvested and washed with PBS. The pellets were resuspendedin Laemmli sample buffer and lysed by boiling.

Sera of patients affected by gastritis and ulcers (provided by A.Ponzetto, hospital “Le Molinette”, Torino, Italy) and sera of patientswith gastric carcinoma (provided by F. Roviello, University of Siena,Italy) were used.

6.1.2. Immunoscreening of the Library

Five hundred thousand plaques of a λgt11 H. pylori DNA expressionlibrary were mixed with 5 ml of a suspension of E. coli strain Y1090grown O/N in LB with 0.2% Maltose and 10 mM MgSO₄, and resuspended in 10mM MgSO₄ at 0.5 O.D. After 10 minutes incubation at 37° C., 75 ml ofmelted TopAgarose were poured in the bacterial/phage mix and the wholewas plated on BBL plates (50,000 plaques/plate). After 3.5 hrsincubation of the plated library at 42° C., nitrocellulose filters(Schleicher and Schuell, Dassel, Germany), previously wet with 10 mMIPTG, were set on plates and incubation was prolonged for 3.5 hrs at 37°C. and then O/N at 4° C. Lifted filters with lambda proteins were rinsein PBS, and saturated in 5% nonfat dried milk dissolved in TBST (10 mMTRIS pH 8, 100 mM NaCl, 5M MgCl₂) for 20′. The first hybridization stepwas performed with the sera of patients; to develop and visualizepositive plaques we used an anti human Ig antibody alkaline phosphataseconjugated (Cappel, West Chester, Pa.) and the NBT/BCIP kit (Promega,Madison, Wis.) in AP buffer (100 mM Tris pH 9.5, 100 mM NaCl, 5mM MgCl₂)according to the manufacturer instructions.

6.1.3. Recombinant DNA Procedures

Reagents and restriction enzymes used were from Sigma (St. Louis, Mo.)and Boehringer (Mannheim, Germany). Standard techniques were used formolecular cloning, single-stranded DNA purification, transformation inE. coli, radioactive labelling of probes, colony screening of the H.pylori DNA genomic library, Southern blot analysis, PAGE and Westernblot analysis.

6.1.4. DNA Sequence Analysis

The DNA fragments were subcloned in Bluescript SK+ (Stratagene, SanDiego, Calif.). Single-stranded DNA sequencing was performed by using[³³P]adATP (New England Nuclear, Boston, Mass.) and the Sequenase kit(U.S. Biochemical Corp., Cleveland, Ohio) according to the manufacturerinstructions. The sequence was determined in both strands and eachstrand was sequenced, on average, twice. Computer sequence analysis wasperformed using the GCG package.

6.1.5. Recombinant Proteins

MS2 polymerase fusion proteins were produced using the vector pEX34A, aderivative of pEX31. Insert Hp67 (from nucleotide 445 to nucleotide 1402in FIG. 3), and the EcoRI linkers were cloned in frame into the EcoRisite of the vector. In order to confirm the location of the stop codon,the HpG3′ HindIII fragment was cloned in frame into the HindIII site ofpEX34A. Recombinant plasmids were transformed in E. coli K12 H1 Δtrp. Inboth cases after induction, a fusion protein of the expected molecularweight was produced. In the case of the EcoRI/EcoRI fragment, the fusionprotein obtain after induction was electroeluted to immunize rabbitsusing standard protocols.

6.2. Results

6.2.1. Screening of an Expression Library and Cloning of HE. pylori hsp

In order to find a serum suitable for the screening of an H. pylori DNAexpression library, sonicated extracts of H. pylori strain CCUG 17874were tested in Western blot analysis against sera of patients affectedby different forms of gastritis. The pattern of antigen recognition bydifferent sera was variable, probably due to differences in theindividual immune response as well as to the differences in the antigensexpressed by the strains involved in the infection.

Serum N°19 was selected to screen a λgt11 H. pylori DNA expressionlibrary to identify H. pylori specific antigens, expressed in vivoduring bacterial growth. Following screening of the library with thisserum, many positive clones were isolated and characterized. Thenucleotide sequence of one of these, called Hp67, revealed anopen-reading frame of 958 base-pairs, coding for a protein with highhomology to the hsp6o family of heat-shock proteins, Ellis, Nature358:191-92 (1992). In order to obtain the entire coding region, we usedfragment Hp67 as a probe on Southern blot analysis of H. pylori DNAdigested with different restriction enzymes. Probe Hp67 recognized twoHindIII bands of approximately 800 and 1000 base-pairs, respectively. Agenomic H. pylori library of HindIII-digested DNA was screened withprobe Hp67 and two positive clones (HpG5′ and HpG3′) of the expectedmolecular weight were obtained. E. coli containing plasmids pHp60G2(approximately nucleotides 1 to 829) and pHp60G5 (approximatelynucleotides 824 to 1838) were deposited with the American Type CultureCollection (ATCC).

6.3. Sequence Analysis

The nucleotide sequence analysis revealed an open-reading frame of 1638base-pairs, with a putative ribosome binding site 6 base-pairs upstreamthe starting ATG. FIG. 3 shows the nucleotide and amino acid sequencesof H. pylori hsp. The putative ribosome-binding and the internal HindIIIsite are underlined. Cytosine in position 445 and guanine in position1402 are the first and last nucleotide, respectively, in fragment Hp67.Thymine 1772 was identified as the last putative nucleotide transcribedusing an algorithm for the localization of factor-independent terminatorregions. The open-reading frame encoded for a protein of 546 aminoacids, with a predicted molecular weight of 58.3 KDa and a predicted pIof 5.37. The codon preference of this gene is in agreement with the H.pylori codon usage.

The analysis of the hydrophylicity profiles revealed a protein mostlyhydrophilic, without a predicted leader peptide or other transmembranedomains. The amino terminal sequence showed 100% homology to thesequence of 30 amino acids determined by Dunn et al., Infect. Immun.60:1946-51 (1992) on the purified protein and differed by only on reside(Ser42 instead of Lys) from the sequence of 44 amino acids published byEvans et al, Infect. Immun. 60:2125-27 (1992). (Evans et al., 1992). TheN-terminal sequence of the mature hsp protein did not contain thestarting methionine, indicating that this had been removed aftertranslation.

6.4. Homology with hsp60 Family

The amino acid sequence analysis showed a very strong homology with thefamily of heat-shock proteins hsp60, whose members are present in everyliving organism. Based on the degree of homology between hsp60 proteinsof different species, H. pylori hsp belongs to the subgroup of hsp60proteins of Gram negative bacteria; however, the degree of homology tothe other proteins of the hsp60 family is very high (at least 54%identity).

The homology of the H. pylori hsp with other heat shock proteins isfully exemplified in FIG. 3. The H. pylori hsp or one or more functionalimmunostimulatory domains thereof may be conjugated to anoligosaccharide or polysaccharide using the procedures of Example 1above to produce conjugate compounds according to the invention.

6.5. Expression of Recombinant Proteins and Production of a PolyclonalAntiserum

The inserts of clone Hp67 and of clone HpG3′ were subcloned in theexpression vector pEX34A in order to express these open-reading framesfused to the aminoterminus of the MS2 polymerase. The clones producedrecombinant proteins of the expected size and were recognized by thehuman serum used for the initial screening. The fused protein derivedfrom clone Hp67 was electroeluted and used to immunize rabbits in orderto obtain anti-hsp specific polyclonal antisera. The antiserum obtainedrecognized both fusion proteins, and a protein of 58 KDa on whole-cellextracts of several strains of H. pylori tested, including aurease-negative strain and noncytotoxic strains.

Hsp has been shown to be expressed by all the H. pylori strains testedand its expression is not associated with the presence of the urease orwith the cytotoxicity. The protein recognized by the anti-hsp antiserumwas found in the water soluble extracts of H. pylori and copurified withthe urease subunits. This suggests a weak association of this proteinwith the outer bacterial membrane. Thus, hsp can be described asurease-associated and surface exposed. The cellular surface localizationis surprising as most of the hsp homologous proteins are localized inthe cytoplasm or in mitochondria and plastids. The absence of a leaderpeptide in hsp suggests that this is either exported to the membrane bya peculiar export system, or that the protein is released from thecytoplasm and is passively adsorbed by the bacterial membrane afterdeath of the bacterium.

7. DEPOSIT OF BIOLOGICAL MATERIALS

The following materials were deposited on Dec. 15, 1992 and Jan. 22,1993 by Biocine Sclavo, S.p.A., the assignee of the present invention,with the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., phone (301) 231-5519, under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor Purposes of Patent Procedure.

ATCC No. 69155 E. coli TG1 containing the plasmid pHp60G2

ATCC No. 69156 E. coli TG1 containing the plasmid pHp605

These deposits are provided as convenience to those of skill in the art,and are not an admission that a deposit is required under 35 U.S.C. §112or any equivalent provision in any one of the designated states herein.The nucleic acid sequences of these deposits, as well as the amino acidsequences of the polypeptides encoded thereby, are incorporated hereinby reference and should be referred to in the event of any error in thesequences described herein as compared with the sequences of thedeposits. A licence may be required to make, use, or sell the depositedmaterials, and no such license is granted hereby.

What is claimed is:
 1. A conjugate compound comprising a portion of atleast 11 to 15 amino acid residues of a heat shock protein selected fromthe group consisting of M. bovis BCG GroEL-type 65 kDa heat shockprotein and recombinant M. tuberculosis DnaK-type 70 kDa heat shockprotein, wherein said heat shock protein portion includes at least oneimmunostimulatory domain, said conjugate compound also comprising atleast one capsular oligosaccharide or capsular polysaccharide, orimmunogenic portion thereof.
 2. A conjugate compound according to claim1 comprising oligosaccharides of the Meningicocci C (MenC) group and aportion of a heat shock protein.
 3. A process for producing a conjugatecompound according to claim 1 which comprises covalently bonding saidheat shock protein portion including at least one immunostimulatorydomain to at least one oligosaccharide or polysaccharide.
 4. A method ofinducing an immune response comprising administering an immunologicallyeffective amount of a conjugate compound according to claim
 1. 5. Aconjugate compound comprising a portion of at least 11 to 15 amino acidresidues of a heat shock protein selected from the group consisting ofM. bovis BCG GroEL-type 65 kDa heat shock protein and recombinant M.tuberculosis DnaK-type 70 kDa heat shock protein, wherein said heatshock protein portion includes at least one immunostimulatory domain,said conjugate compound also comprising at least one capsularoligosaccharide or capsular polysaccharide, or immunogenic portionthereof from a bacteria selected from the group consisting ofHemophilus, Salmonella, Streptococcus, and Shigella.
 6. A conjugatecompound comprising at least one heat shock protein from H. pylori ofabout 54-62 kDa, wherein said heat shock protein includes at least oneimmunostimulatory domain, said conjugate compound also comprising atleast one capsular oligosacchalide or capsular polysaccharide, orimmunogenic portion thereof.
 7. The conjugate compound of claim 6comprising the heat shock protein from H. pylori of about 54-62 kDa, ora portion thereof, wherein said heat shock protein includes at least oneimmunostimulatory domain, said conjugate compound also comprising atleast one capsular oligosaccharide or capsular polysaccharide, orimmunogenic portion thereof from a bacteria selected from the groupconsisting of Hemophilus, Salmonella, Streptococcus, and Shigella. 8.The conjugate compound of claim 6 comprising the heat shock protein fromH. pylori of about 54-62 kDa, wherein said heat shock protein includesat least one immunostimulatory domain, said conjugate compound alsocomprising at least one capsular oligosaccharide or immunogenic portionthereof from a bacteria selected from the group consisting ofHemophilus, Salmonella, Streptococcus, and Shigella.
 9. A conjugatecompound comprising a portion of a heat shock protein selected from thegroup consisting of M. bovis BCG GroEL-type 65 kDa heat shock proteinand recombinant M. tuberculosis DnaK-type 70 kDa heat shock protein,wherein said portion of said heat shock protein includes animmunostimulatory domain wherein said portion includes one or more ofthe regions underlined in SEQ ID NO:5 of FIG. 2, said conjugate compoundalso comprising at least one capsular oligosaccharide or capsularpolysaccharide, or immunogenic portion thereof.
 10. A conjugate compoundcomprising a portion of a heat shock protein selected from the groupconsisting of M. bovis BCG GroEL-type 65 kDa heat shock protein andrecombinant M. tuberculosis DnaK-type 70 kDa heat shock protein, whereinsaid portion of said heat shock protein includes an immunostimulatorydomain wherein said portion includes one or more of the regionsunderlined in SEQ ID NO:5 of FIG. 2, said conjugate compound alsocomprising at least one capsular oligosaccharide or capsularpolysaccharide, or immunogenic portion thereof from a bacteria selectedfrom the group consisting of Hemophilus, Salmonella, Streptococcus, andShigella.
 11. The conjugate compound of any one of claims 9 or 10wherein said one of said regions comprises: amino acid residues 27 to38, amino acid residues 41 to 48, amino acid residues 50 to 58, aminoacid residues 60 to 88, amino acid residues 90 to 94, amino acidresidues 98 to 107, amino acid residues 109 to 135, amino acid residues141 to 147, amino acid residues 152 to 189, amino acid residues 202 to251, or amino acid residues 254 to 272 of SEQ ID NO:5.